The structure of petromyzestrosterol, a pheromonal steroid of the sea lamprey, was verified by total syntheses of both C14-epimers of its 3-O-methyl derivative. The key features of our synthesis involve (1) a highly stereoselective Mizoroki-Heck reaction to unite the A- and CD-ring segments and (2) Friedel-Crafts-type cyclodehydration to construct the B-ring. Petromyzestrosterol is concluded to bear an α-configured C14 hydroxy group based on a comparison of NMR data of both the synthesized C14-epimers of the 3-O-methyl derivative with those of the natural petromyzestrosterol. The downfield shifts of C9 and C12 via the γ-gauche effect in the 14β-isomer would enable the structural elucidation of C14 in the 14-hydroxy estrogenic steroids.